Mutations will be introduced by enzymatic and chemical methods into localized regions at the left end of the Adenovirus 5 genome. In separate experiments mutagenesis will be directed to the 5' and 3' ends of the early region encoded there (early region 1, the transforming region of Ad5) and into areas where sequence is joined in spliced mRNAs from this early region. Host range mutants generated by these methods will be selected by their ability to grow on 293 cells, a human cell line transformed by Ad5 which expresses early region 1 functions, and their inability to grow on HeLa cells. mRNA- mutants will be isolated by screening the generated host range mutants with the sensitive and simple S1 mapping method to identify mutants which fail to induce early mRNAs in HeLa cells. mRNA- mutants will be mapped by marker rescue and sequenced. An adaption of the S1 mapping procedure to the analysis of pulse labeled transcripts will be used to characterize the biochemical defect in these mutants. By extensively mutagenizing localized regions throughout early region 1, it should be possible to identify all steps in mRNA production which require specific DNA and RNA sequences and to define these sequences. Determining the sequence of mutations which revert the mRNA- mutants will yield insight into the mechanisms of these steps. The host range mutants obtained in these studies will be placed into complementation groups and representatives of each complementation group will be assayed for their ability to transform rat embryo and hamster embryo cells. Such studies will define the Ad5 genes required for transformation by Ad5.